The phylogeography of the eastern honeybee (Apis cerana) in southern China is poorly understood. In the present study, we investigated phylogenetic relationships among A. cerana populations on Hainan island and southern mainland China. Analyses were based on the partial cytochrome oxidase subunit 1 gene of mitochondrial DNA and A. cerana samples representing 12 geographic populations. We identified 57 novel haplotypes (GenBank accession numbers: KC175488-KC175544) within our sampled populations and inferred two deeply divergent intraspecific lineages when previously published sequences were included, which could be further divided into six sublineages. We also found a significant phylogeographic structure within lineage A. Our results reveal that A. cerana populations on Hainan island and southern mainland China have undergone a long period of differentiation in isolation followed by historical demographic expansion. Although gene flow now occurs between these locations, gene flow has occurred over comparatively a short time period, and is likely to be the result of anthropogenic effects.